Enhanced virulence mediated by the murine coronavirus, mouse hepatitis virus strain JHM, is associated with a glycine at residue 310 of the spike glycoprotein

Abstract

The coronavirus, mouse hepatitis virus strain JHM, causes acute and chronic neurological diseases in rodents. Here we demonstrate that two closely related virus variants, both of which cause acute encephalitis in susceptible strains of mice, cause markedly different diseases if mice are protected with a suboptimal amount of an anti-JHM neutralizing antibody. One strain, JHM.SD, caused acute encephalitis, while infection with JHM.IA resulted in no acute disease. Using recombinant virus technology, we found that the differences between the two viruses mapped to the spike (S) glycoprotein and that the two S proteins differed at four amino acids. By engineering viruses that differed by only one amino acid, we identified a serine-to-glycine change at position 310 of the S protein (S310G) that recapitulated the more neurovirulent phenotype. The increased neurovirulence mediated by the virus encoding glycine at position S310 was not associated with a different tropism within the central nervous system (CNS) but was associated with increased lateral spread in the CNS, leading to significantly higher brain viral titers. In vitro studies revealed that S310G was associated with decreased S1-S2 stability and with enhanced ability to mediate infection of cells lacking the primary receptor for JHM ("receptor-independent spread"). These enhanced fusogenic properties of viruses encoding a glycine at position 310 of the S protein may contribute to spread within the CNS, a tissue in which expression of conventional JHM receptors is low.

FIG. 1.

Schematic of recombinant viruses. (A) Interspecies chimeric virus fMHV-JHM B3b. fMHV-JHM carries the region from gene 1 through the 5′ end of HE from JHM.IA, the 3′ end of HE from MHV-A59, the ectodomain of FIPV gene 3, the transmembrane domain and endodomain of gene 3 from MHV-A59, and the region from gene 4 through the 3′ untranslated region (3′ UTR) from MHV-A59 (45). This chimeric virus infects only feline cells. (B) RNA transcribed from pJHM. As an example, construction of rJHM.IA is shown. Donor RNA (8 kb) was transcribed in vitro as described in Materials and Methods. pJHM.IA carries sequences entirely derived from JHM.IA. (C) Construction of rJHM.IA. fMHV-JHM B3b-infected feline cells were transfected with donor RNA transcribed from pJHM.IA to create rJHM.IA. (D) Donor RNA from pJHM.SD was used to create rJHM.SD. Point mutations were introduced into pJHM.IA to create the recombinant viruses listed. The sources of genomic or donor RNA segments are indicated by open rectangles for JHM.IA, hatched rectangles for FIPV, and shaded rectangles for MHV-A59. S1-S2 cleavage occurs between nt 2307 and 2308.

FIG. 2.

Virulence of spike variants. Suckling mice were infected intranasally with the indicated viruses and nursed by dams actively immunized with infectious JHM.IA (A) or passively immunized with a cocktail of two anti-JHM neutralizing antibodies (B) as described in Materials and Methods. All pups were monitored daily for signs consistent with acute encephalitis (hunching, ruffled fur, lethargy). The fraction surviving at each day p.i. is shown. Each virus was used to infect five to eight mice. Symbols: ○, JHM.IA; •, JHM.SD; □, rJHM.IA; ▪, rJHM.SD; ▴, rJIA-S.T61A; ◊, rJIA-S.I154T; ⧫, rJIA-S.S310G; ×, rJIA-S.L539F. Note that only JHM.IA, JHM.SD, rJHM.SD, rJHM.IA, and rJIA-S.S310G are analyzed in panel A.

FIG. 3.

Neutralization of JHM.IA and JHM.SD by maternal serum. Dams were immunized three to five times with infectious JHM.IA. Sera were prepared 3 weeks after the last immunization and treated at 56°C for 30 min. Dilutions of sera were incubated with an equal volume of the indicated virus for 30 min on ice. Residual virus titers were determined by a plaque assay on HeLa-MHVR cells as described in Materials and Methods. Representative curves with serum from an individual dam are shown. Sera from five dams were analyzed in these experiments with similar results. Symbols: ⋄, JHM.IA; □, JHM.SD. Ab, antibody.

FIG. 4.

Kinetics of virus production in 17Cl-1 cells. Cells were infected with the indicated viruses at a multiplicity of 1 PFU per cell. Cells and supernatants were harvested at the indicated times and freeze-thawed. Titers were measured by plaque assay on HeLa-MHVR cells. Symbols: □, JHM.IA; ○, JHM.SD; ▪, rJHM.IA; •, rJHM.SD.

FIG. 5.

Enhanced spread within the CNS by viruses encoding a Gly at position 310 of the S protein. Suckling mice were infected intranasally at the age of 10 days with either JHM.SD (A), JHM.IA (B), rJHM.SD (C), rJHM.IA (D), rJIA-S.T61A (E), rJIA-S.I154T (F), rJIA-S.S310G (G), or rJIA-S.L539F (H). Five days p.i., brains were harvested and fixed as described in Materials and Methods. Viral antigen was detected with the anti-N MAb 5B188.2 and counterstained with hematoxylin.

FIG. 6.

Effects of the JHM.IA mutations on S1 shedding from virions. 17Cl-1 cells infected with rJHM.SD (A), rJHM.IA (B), or rJ.IA.S.S310G (C) were pulse-labeled for 30 min with Tran³⁵S-label and then chased in nonradioactive medium for the indicated times. Media were then collected, virions were pelleted by ultracentrifugation, and S1 fragments which remained soluble were immunoprecipitated onto NCEACAM-Fc beads (16). Radioactive proteins were separated by electrophoresis and visualized by fluorography.

FIG. 7.

Thermostability of spike variants. Cell-free virus was incubated in solutions at a pH of 6.0 (A), 7.0 (B), or 8.0 (C) at 37°C. Aliquots were removed at the indicated time points and titered on HeLa-MHVR cells. The fraction of virus surviving at each time point is shown. Symbols: (□), JHM-IA; ○, JHM-SD; ▪, rJHM.IA; •, rJHM.SD; +, rJIA-S.S310G. Data are representative of four independent experiments.

FIG. 8.

Effect of a Gly at position 310 of the S protein on receptor-independent spread. 17Cl-1 cells (JHM permissive) infected with rJHM.IA (A, D, and G), rJHM.SD (B, E, and H), or rJIA.S.S310G (C, F, and I) were overlaid onto CFSE-labeled uninfected 17Cl-1 cells (A through C) or BHK cells (non-JHM permissive) (D through I) at a 1:50 ratio as described in Materials and Methods. At 20 h p.i., cells were fixed, and viral antigen was detected by sequential treatments with an anti-N antibody and a Texas red-conjugated goat anti-mouse antibody. Cells were visualized by fluorescent microscopy. Green fluorescence, CFSE labeling; red fluorescence, Texas red labeling. Only CSFE-labeled cells are shown in panels A through C.